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Objective:To investigate the protective effect of pine pollen on lipopolysaccharide (LPS)-induced learning and memory impairments in mice and the underlying mechanism.Methods:Sixty mice were randomly divided into four groups ( n = 15/group): normal control, model, low-dose pine pollen (500 mg/kg) and high-dose pine pollen (1 000 mg/kg). Mouse models of learning and memory impairment were established by lateral ventricle injection of LPS. The learning and memory abilities of mice were determined by the Morris water maze test. Superoxide dismutase (SOD) activity and glutathione (GSH) and malondialdehyde (MDA) levels in the hippocampus of mice were measured. Interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), dopamine (DA), and norepinephrine (NE) levels in the hippocampus were also determined. Results:The latency in the passive avoidance test in the model group was significantly shorter than that in the normal control group [(134.80 ± 33.89) s vs. (282.20 ± 17.43) s, t = 4.23, P < 0.01]. The number of errors in the model group was significantly higher than that in the normal control group [(4.00 ± 1.58) vs. (1.20 ± 1.30) times, t = 2.85, P < 0.01]. The latency in the passive avoidance test in the low-dose pine pollen (500 mg/kg) and high-dose pine pollen (1000 mg/kg) groups was significantly longer than that in the normal control group [(189.40 ± 27.21) s or (213.40 ± 21.26) s vs. (134.80 ± 33.89) s, t = 3.21, 4.38, all P < 0.05]. The number of errors in the low-dose pine pollen (500 mg/kg) and high-dose pine pollen (1 000 mg/kg) groups was significantly lower than that in the normal control group [(1.60 ± 1.44) times or (1.40 ± 1.44) times vs. (4.00 ± 1.58) times, t = 5.12, 6.42, both P < 0.05]. SOD activity and GSH, DA and NE levels in the hippocampus in the model group were significantly decreased compared with the normal control group [SOD: (7.59 ± 1.77) kU/g vs. (39.90 ± 6.37) kU/g; GSH: (3.49 ± 0.13) mmol/g vs. (6.37 ± 0.14) mmol/g; DA: (418.42 ± 2.57) ng/L vs. (586.37 ± 3.64) ng/L; NE: (187.20 ± 5.41) ng/L vs. (298.42 ± 2.32) ng/L, t = 3.67, 8.23, 2.23, 3.65, all P < 0.05]. MDA, IL-6 and TNF-α levels in the hippocampus in the normal control group were significantly higher than those in the model group [MDA: (8.79 ± 0.82) mmol/g vs. (2.62 ± 0.16) mmol/g, IL-6: (48.07 ± 5.56) ng/L vs. (18.76 ± 1.42) ng/L, TNF-α: (87.20 ± 4.31) ng/L vs. (22.42 ± 3.39) ng/L, t = 7.45, 2.67, 4.35, P < 0.05 or P < 0.01]. SOD activity, GSH, DA and NE levels in the hippocampus in the low-dose pine pollen (500 mg/kg) and high-dose pine pollen (1 000 mg/kg) groups were significantly higher than those in the model group [SOD: (18.80 ± 2.39) kU/g, (28.70 ± 2.36) kU/g vs. (7.59 ± 1.77) kU/g, GSH: (5.04 ± 0.36) mmol/g, (5.45 ± 0.17) mmol/g vs. (3.49 ± 0.13) mmol/g, DA: (488.37 ± 3.46) ng/L, (506.29 ± 5.72) ng/L vs. (418.42 ± 2.57) ng/L, NE: (225.65 ± 3.72) ng/L, (239.76 ± 5.58) ng/L vs. (187.20 ± 5.41) ng/L, t = 4.56 or 6.71, t = 4.65 or 5.32, t = 4.73 or 6.72, t = 3.84 or 5.63, P < 0.05 or P < 0.01]. MDA, IL-6 and TNF-α levels in the hippocampus in the low-dose pine pollen (500 mg/kg) and high-dose pine pollen (1 000 mg/kg) groups were significantly lower than those in the model group [MDA: (5.72 ± 0.47) mmol/g, (3.77 ± 0.23) mmol /g vs. (8.79 ± 0.82) mmol/g, IL-6: (28.42 ± 3.54) ng/L, (23.43 ± 5.62) ng/L vs. (48.07 ± 5.56) ng/L, TNF-α: (48.87 ± 4.82) ng/L, (39.65 ± 6.69) ng/L vs. (87.20 ± 4.31) ng/L, t = 6.31 or 7.28, t = 3.46 or 6.31, t = 4.28 or 3.57, P < 0.05 or P < 0.01]. Conclusion:Pine pollen can improve LPS-induced learning and memory impairments possibly through up-regulating the levels of monoamine neurotransmitters DA and NE and inhibiting the levels of oxidative stress and inflammatory reaction in the hippocampus of mice.
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The development of sweat glands is a very complex biological process, which involves many factors. In this study, the correlation between epidermal growth factor (EGF), matrix metalloproteinases (MMP 2,MMP 7) and development of sweat glands in human embryos was explored. Furthermore, the mechanism(s) underlying the induction of epidermal stem cells into sweat gland cells was elucidated. Skin biospies of human embryos obtained from spontaneous abortions at different gestational ages from 11 to 31 weeks were used. The dynamical expression of EGF, MMP 2, MMP 7 and keratin 7(K7) in developing sweat gland cells or extracellular stroma surrounding the sweat gland cells were examined with S P immunohistochemical methods. The localization of the cellular sources of MMP 2 and MMP 7 was examined with in situ hybridization. The results showed that at 14 20 wk of gestation, a gradual increase of EGF immunoreactivity was observed not only in developing sweat gland buds but also in extracellular stroma surrounding the buds,and the expression intensity peaked at 20～22 wk of gestational age. All mRNA positive buds or cells in developing sweat glands contained corresponding immunoreactive proteins. The immunostaining for K7 appeared in early sweat gland buds at 14～16wk of gestation, and from then on, K7 was concentrated in developing sweat gland cords or cells. It is suggested that the morphogenesis of sweat gland in human fetal skin begins at 14～16wk of gestational age, and essentially completes by 24wk. There is a close relationship among EGF ,extracellular matrix remodeling and morphogenesis of sweat glands, and EGF is one of the inducers in the development and maturity of sweat gland buds or cells.
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To investigate the culture method for epidermal stem cell in vitro. Rat skin was peeled from the subcutaneous tissue gently, and cut into small pieces, then incubated for 24h in 0 25%trypsin in a 4℃ shaker. The epidermis was separated from the dermis, and shaken for 10min in 0 25% trypsin at 37℃ to dissociate into single cells. Digestion was terminated by the addition of culture medium +10% serum, and the cells were gently centrifuged and resuspended in the culture medium, which constituted EMEM without calcium, supplemented with 0 05mmol/L CaCl 2 , 9% chelexed FBS, 4ng/ml EGF, 0 5?g/ml gentamicin, and 50% fibroblast conditioned medium (CM ). The CM ( EMEM without calcium, 0 05mmol/L CaCl 2 , 9% chelexed FBS, 0 5?g/ml gentamicin ) was collected from freshly isolated primary neonate fibroblast cultures after 48h, which were passed through a filter with 0 45?m pores, and stored at -20℃ for use. 1?10 6 /ml epidermal cells were incubated for 10～15min at 37℃ on dishes coated with collagen Ⅳ, then the nonadherent cells were rinsed off. The adherent cells were grown to confluency. Cultures were subcultured in a solution of TGG for 5～10min. Once free of the dish, cells were centrifuged, resuspended in fresh medium, and then cultured at 1?10 5 /ml. Cultures were observed for colony formation under a phase contrast microscope, and the structure of the rapidly adherent cells was observed under electron microscopy; the expression of ? 1 integrin and K19 was detected in the rapidly adherent cells and colony cells with SP immunohistochemical methods. The results showed that the cells selected by rapid adherence to collagen type Ⅳ formed large colonies at 7 days, and showed a im mature feature under electron microscopy, manifesting ? 1 integrin and K19 expression. It is suggested that epidermal stem cells could be cultured in vitro .
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To study the apoptotic regularity of peripheral lymphocytes in radiation compound wound healing and explore their relationship to radiation delayed wound healing, apoptosis and Bax and Bcl 2 proteins were estimated by in situ terminal labeling (TUNEL) and immunohistochemical method of alkaline phosphatase, respectively. White blood cells in radiation compound wound(RW) group decreased and reached the lowest 3 days after wound, it was still lower in RW group than in simple wound(SW) group 5 days after wound; apoptosis of peripheral lymphocytes of both groups increased, however, the lymphocyte apoptosis in RW group was higher than that in SW from beginning to end in the whole process of wound healing; the expression of Bax protein of lymphocyte increased at different times after wound, and the expression in RW group was obviously higher than that in SW group, while Bcl 2 showed an opposite tendency. It is suggested that reduction of white blood cells and increment of lymphocyte apoptosis are the major causes of radiation delayed wound healing; Bax and Bcl 2 proteins take part in the regulation of lymphocyte apoptosis in wound healing.